Fingolimod synergizes with anti-PD-1 radioimmunotherapy in an experimental multiple sclerosis model through enhanced lymph node retention and CD8+ T cell depletion

Connor Frank^1*Wojciech Dawicki²Michael C. Levin^3,4Ekaterina Dadachova^1*

• ¹College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
• ²Department of Biochemistry, Microbiology and Immunology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
• ³Division of Neurology, Department of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
• ⁴Department of Anatomy, Physiology and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Multiple sclerosis (MS) is a chronic, neurodegenerative autoimmune disease caused by inappropriate activation of the immune system that targets central nervous system antigens, such as myelin. Some MS therapies control the disease through the depletion of CD20 + B cells and sequestration of T-lymphocytes in lymph nodes. A targeted approach to deplete only activated T lymphocytes, a key player in disease progression, would be optimal. The programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed on exhausted and recently activated T-cells. We have previously reported success with [ 177 Lu]Lu-anti-PD-1 radioimmunotherapy (RIT) as a monotherapy in experimental autoimmune encephalomyelitis (EAE), the most common preclinical model of MS. Here we present the synergistic effect of fingolimod (FTY720/Gilenya™) in combination with [177Lu]Lu-aPD-1 in EAE. Methods: [177Lu]Lu-aPD-1 and [111In]In-aPD-1 were prepared by conjugating a commercial anti-murine PD-1 antibody with DOTA bifunctional chelating agent. [177Lu]Lu-aPD-1 ± FTY720 therapy was initiated at EAE symptom onset in MOG 35-55 immunized C57Bl/6 mice. ImmunoSPECT/CT imaging utilizing [111In]In-aPD-1 was performed ± FTY720 administration at symptom onset in EAE mice. Flow cytometry on mononuclear cells isolated from RIT ± FTY720 was performed on day 3 post treatment. Results: [177Lu]Lu-aPD-1 in combination with FTY720 administration significantly reduced long-term paralysis in EAE mice. ImmunoSPECT/CT imaging demonstrated enhanced lymph node retention of [111In]In-aPD-1 antibody in conjugation with FTY720 corresponding to presence of activated lymphocytes in lymph nodes. Flow cytometry performed on isolated spinal cord mononuclear cells demonstrated significantly reduced CD8+ T cell counts in spinal cords of [177Lu]Lu-aPD-1 and [177Lu]Lu-aPD-1 + FTY720 treated animals. Immunofluorescent microscopy on thoracic spinal cord sections from treated animals demonstrated reduced demyelination and moderate infiltration of CD3+ lymphocytes in [177Lu]Lu-aPD-1 + FTY720 group at study termination. Conclusion: [177Lu]Lu-aPD-1 targeted radioimmunotherapy was synergistically enhanced utilizing the approved MS therapeutic FTY720 through increased lymph node retention and irradiation of activated T lymphocytes. This work paves the way for application of PD-1+ T cell targeted radioimmunotherapy as a potential theranostic agent for MS.